Project Summary Glioblastoma (GBM) is the most common primary brain malignancy and carries a uniformly poor prognosis. Immunotherapy has the potential to improve outcomes by generating a highly specific anti-tumor response, but efficacy has been limited by the immunosuppressive systemic and intratumoral microenvironment encountered in GBM patients. Immunotherapy for GBM relies on induction of tumor specific CD8+ cytolytic T cells (CTLs). CTL pro-inflammatory activity is balanced by a number of anti-inflammatory immune populations, including inducible regulatory T cells (iTregs) and myeloid derived suppressor cells (MDSC). The relative activity of CTLs versus anti-inflammatory immune effectors determines the capacity of the immune system to control tumor growth. iTregs are known to be activated during education and expansion of effector T cells. Data suggests that binding of PD-1 on nave CD4+ T cells to it's ligand, PD-L1, can substantially increase the expansion of Tregs during activation. Expression of PD-L1 on tumor cells and circulating myeloid cells has been demonstrated in GBM, and may be a significant factor in the expansion of iTregs and the development of immunoresistance. Preliminary evidence indicates that (1) Tregs are significantly expanded in peripheral blood and tumors of patients with GBM, (2) binding of PD-1 to PD-L1 during T cell activation increases iTreg expansion, (3) PD-L1 is upregulated on tumor cells and myeloid cells in GBM patients, and (4) PD-L1 expression on myeloid cells correlates with survival in GBM patients receiving vaccine immunotherapy. Given these relationships, we hypothesize that tumor-induced PD-L1 expression on myeloid cells in GBM contributes to expansion of iTregs. Further, inhibition of PD-1 has known to impair T regulatory cell conversion. In this proposal, we will investigate the relationship with PD-L1 expression of GBM and regulatory T cell expansion. Moreover, we will explore how these variables affect patient survival and functional immunosuppression. The second aim will focus on identifying key signaling pathways involved in Treg expansion in addition to investigating how PD-L1 upregulates T reg expression in nave T cell population. The third proposed aim will evaluate the role of PD-1 inhibition on Treg population along with tumor growth and survival in murine GBM models. Through these experiments, we hope to gain an understanding of the mechanisms driving immunosuppression to develop novel strategies to increase the efficacy of immunotherapeutic approaches for treating GBM.